Method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network

ABSTRACT

A method of preparing a supramolecular complex containing at least one therapeutic agent and a multi-dimensional polymer network is described. A supramolecular complex prepared by a method of the invention is described. A method of treatment by administering a therapeutically effective amount of a supramolecular complex of the invention is also described. Such a supramolecular complex may be used as a delivery vehicle for various therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/075,977, filed Mar. 13, 2008, which is a continuation of U.S. patentapplication Ser. No. 09/453,707, filed Dec. 3, 1999, now U.S. Pat. No.7,375,096, which claims the benefit of U.S. Provisional Application Nos.60/110,847, filed Dec. 4, 1998, and 60/127,856, filed Apr. 5, 1999, allof which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a method of preparing a supramolecular complexcontaining at least one therapeutic agent (e.g. DNA) and amulti-dimensional polymer network. Such a supramolecular complex may beused as a delivery vehicle of a therapeutic agent.

BACKGROUND OF THE INVENTION

Cyclodextrins are cyclic polysaccharides containing naturally occurringD(+)-glucopyranose units in an α-(1,4) linkage. The most commoncyclodextrins are alpha (α)-cyclodextrins, beta (β)-cyclodextrins andgamma (γ)-cyclodextrins which contain, respectively, six, seven or eightglucopyranose units. Structurally, the cyclic nature of a cyclodextrinforms a torus or donut-like shape having an inner apolar or hydrophobiccavity, the secondary hydroxyl groups situated on one side of thecyclodextrin torus and the primary hydroxyl groups situated on theother. Thus, using (β)-cyclodextrin as an example, a cyclodextrin isoften represented schematically as follows:

The side on which the secondary hydroxyl groups are located has a widerdiameter than the side on which the primary hydroxyl groups are located.The hydrophobic nature of the cyclodextrin inner cavity allows for theinclusion of a variety of compounds. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996);T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al.,Applied Spectroscopy, 46:652-658 (1992); FR 2 665 169).

Cyclodextrins have been used as a delivery vehicle of varioustherapeutic compounds by forming inclusion complexes with various drugsthat can fit into the hydrophobic cavity of the cyclodextrin or byforming non-covalent association complexes with other biologicallyactive molecules such as oligonucleotides and derivatives thereof. Forexample, U.S. Pat. No. 4,727,064 describes pharmaceutical preparationsconsisting of a drug with substantially low water solubility and anamorphous, water-soluble cyclodextrin-based mixture. The drug forms aninclusion complex with the cyclodextrins of the mixture. In U.S. Pat.No. 5,691,316, a cyclodextrin cellular delivery system foroligonucleotides is described. In such a system, an oligonucleotide isnoncovalently complexed with a cyclodextrin or, alternatively, theoligonucleotide may be covalently bound to adamantine which in turn isnon-covalently associated with a cyclodextrin.

Various cyclodextrin containing polymers and methods of theirpreparation are also known in the art. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996)).A process for producing a polymer containing immobilized cyclodextrin isdescribed in U.S. Pat. No. 5,608,015. According to the process, acyclodextrin derivative is reacted with either an acid halide monomer ofan α,β-unsaturated acid or derivative thereof or with an α,β-unsaturatedacid or derivative thereof having a terminal isocyanate group or aderivative thereof. The cyclodextrin derivative is obtained by reactingcyclodextrin with such compounds as carbonyl halides and acidanhydrides. The resulting polymer contains cyclodextrin units as sidechains off a linear polymer main chain.

U.S. Pat. No. 5,276,088 describes a method of synthesizing cyclodextrinpolymers by either reacting polyvinyl alcohol or cellulose orderivatives thereof with cyclodextrin derivatives or by copolymerizationof a cyclodextrin derivative with vinyl acetate or methyl methacrylate.Again, the resulting cyclodextrin polymer contains a cyclodextrin moietyas a pendant moiety off the main chain of the polymer.

A biodegradable medicinal polymer assembly with supermolecular structureis described in WO 96/09073 A1 and U.S. Pat. No. 5,855,900. The assemblycomprises a number of drug-carrying cyclic compounds prepared by bindinga drug to an α, β or γ-cyclodextrin and then stringing thedrug/cyclodextrin compounds along a linear polymer with thebiodegradable moieties bound to both ends of the polymer. Such anassembly is reportably capable of releasing a drug in response to aspecific biodegradation occurring in a disease. These assemblies arecommonly referred to as “necklace-type” cyclodextrin polymers.

SUMMARY OF THE INVENTION

The invention provides a method of preparing a supramolecular complexcomprising at least one therapeutic agent and a multi-dimensionalpolymer network. According to such a method, at least one therapeuticagent is contacted with at least one polymer to form a composite. Thepolymer of the composite is then treated under conditions sufficient toform a supramolecular complex containing the therapeutic agent and amulti-dimensional polymer network.

The invention also provides a supramolecular complex containing at leastone therapeutic agent and a multi-dimensional polymer network.

The invention further provides a method of treatment by administering atherapeutically effective amount of a supramolecular complex containingat least one therapeutic agent and a multi-dimensional polymer network.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Agarose Gel of Reversible Crosslinking of Branched PEI (25 kD)with DTBP.

FIG. 2A. Transfection with CD-DMS, CD-DMA, and CD-DMP.

FIG. 2B. Toxicity of CD-DMS, CD-DMA, and CD-DMP.

FIG. 2C. Transfection to BHK-21 Cells (serum free) with CD-DMS andCD-DTBP.

FIG. 2D. Toxicity of CD-DMS and CD-DTBP with BHK-21 cells (serum free).

FIG. 3A. Transfection of C6 and C9 Diamine-DMS Copolymers.

FIG. 3B. Toxicity of C6 and C9 Diamine-DMS Copolymers.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method of preparing a supramolecular complexcontaining at least one therapeutic agent and a multi-dimensionalpolymer network. According to a method of the invention, at least onetherapeutic agent is contacted with at least one polymer to form acomposite and then the polymer of the composite is treated underconditions sufficient to form a supramolecular complex containing thetherapeutic agent and a multi-dimensional polymer network.

A composite of at least one therapeutic agent and at least one polymermay be defined as a combination or integration of at least onetherapeutic agent and at least one polymer, each as described below.According to the invention, a “polymer” is defined as either a singlepolymer molecule (e.g. a single polymer strand or fragment) or as agroup of two or more polymer molecules (e.g. a group of two or morepolymer strands or fragments). Thus, according to the invention, acomposite contains at least one single polymer molecule; at least onegroup of two or more polymer molecules, which may be the same ordifferent; or a mixture of at least one single polymer molecule and atleast one group of two or more polymer molecules, which may be the sameor different. A polymer molecule may be linear or branched. Accordingly,a group of two or more polymer molecules may be linear, branched, or amixture of linear and branched polymers. According to the invention,prior to formation of the composite, the polymer of the composite doesnot exist as a substantially associated structure such as, for example,a polymer gel. However, the polymer as part of the composite, dependingupon the nature of the polymers and the therapeutic agent, may form sucha substantially associated structure. Each polymer of the composite mayfurther contain or may be further modified to contain at least onefunctional group through which association of the polymers of thecomposite may be achieved, as described below.

The composite may be prepared by any suitable means known in the art.For example, the composite may be formed by simply contacting, mixing ordispersing a therapeutic agent with a polymer, each as described herein.A composite may also be prepared by polymerizing monomers, which may bethe same or different, capable of forming a linear or branched polymerin the presence of a therapeutic agent. In a preferred embodiment of theinvention, a composite may be prepared by polymerizing monomers, whichmay be the same or different, capable of forming a linear or branchedpolymer in the presence of a therapeutic agent where the therapeuticagent acts as a template for the polymerization. Trubetskoy et al.,Nucleic Acids Research, Vol. 26, No. 18, pp. 4178-4185 (1998). Thecomposite may be further modified with at least one ligand, as describedbelow. The ligand may be introduced upon or after formation of thecomposite via ligand modification of the therapeutic agent and/or thepolymer of the composite, as described herein. The composite may takeany suitable form and, preferably, is in the form of particles.

According to the invention, the polymer of the composite is treatedunder conditions sufficient to form a supramolecular complex comprisinga therapeutic agent and a multi-dimensional polymer network, each asdescribed herein. “Treatment of the polymer of the composite underconditions sufficient to form a supramolecular complex” may be definedas any suitable reaction condition(s), including the addition ofadditional agents or reactants, that promote association of the polymerof the composite. The polymer, as described above, may be associated viainterpolymer covalent bonds, noncovalent bonds (e.g. ionic bonds), ornoncovalent interactions (e.g. van der Waals interactions). Associationvia intrapolymer covalent bonding, noncovalent bonding, or noncovalentinteractions of the polymer may occur as well. As a result of suchassociation, the polymer of the composite interacts to form amulti-dimensional polymer network. Formation of a multi-dimensionalpolymer network may be determined using spectroscopy. Amulti-dimensional polymer network exhibits different spectrographic data(e.g. infrared spectroscopy, nuclear magnetic resonance (NMR)spectroscopy) than the unassociated polymer of the composite. Inaddition, a multi-dimensional network of at least two polymers has anaverage molecular weight greater than that of the individual polymers ofthe composite.

In a preferred embodiment of the invention, “treatment of the polymer ofthe composite under conditions sufficient to form a supramolecularcomplex” involves crosslinking reaction conditions. For example, if thepolymer of the composite is a single polymer molecule, the polymer maybe reacted with a molecule(s), oligomer(s), or different polymer(s) thatpromotes crosslinking or forms crosslinks such that intrapolymercrosslinking of or actual crosslinking with the single polymer moleculeof the composite results. Similarly, if the polymer of the composite isa group of two or more polymer molecules, the polymer may be reactedwith a molecule(s), oligomer(s), or different polymer(s) that promotescrosslinking or forms crosslinks such that intrapolymer and/orinterpolymer, preferably interpolymer, crosslinking of or actualcrosslinking with the group of two or more polymer molecules of thecomposite results.

The crosslinking agent may be any crosslinking agent known in the art.The crosslinking agent may be any oligomer or polymer (e.g. polyethyleneglycol (PEG) polymer, polyethylene polymer) capable of promotingcrosslinking within or may be actually crosslinking with the polymer ofthe composite. The crosslinking oligomer or polymer may be the same ordifferent as the polymer of the composite. Likewise, the crosslinkingagent may be any suitable molecule capable of crosslinking with thepolymer of the composite.

Examples of crosslinking agents include dihydrazides and dithiols. In apreferred embodiment, the crosslinking agent is a labile group such thata crosslinked multi-dimensional polymer network may be uncrosslinked asdesired. A mixture of different crosslinking agents may also be used.The different crosslinking agents may exhibit varying degrees oflability. Accordingly, the advantage of directed bioavailability (e.g.as in “timed release” formulations) may be achieved. Examples ofsuitable crosslinking agents include, but are not limited to, adipicacid dihydrazide, polyethylene glycol 600 (PEG₆₀₀) dihydrazide, dimethyl3,3′-dithiobispropionimidate (DTBP), dithiobis(succinimidyl propionate)(DSP), disuccinimidyl suberate (DSS), and dimethylsuberimidate (DMS).The crosslinking agent may be further modified with at least one ligandas described herein.

“Treatment of the polymer of the composite under conditions sufficientto form a supramolecular complex” may also include suitable reactionconditions that promote the crosslinking of functional groups found onthe polymer of the composite such that association via a new bond orinteraction, as described above, results. The functional group may beany functional group known in the art which forms a new bond orinteraction, as described above, under crosslinking reaction conditions.In a preferred embodiment of the invention, the polymer of the compositeis functionalized with at least two thiol groups or may be modified tobe functionalized with at least two thiol groups, which underappropriate oxidation conditions react to form a disulfide linkage. Athiol-functionalized polymer may be prepared by means known in the artincluding, for example, the addition of a thiolating reagent (e.g.Traut's Reagent, commercially available from Pierce Chemical Company,Rockford, Ill.). A thiol-functionalized polymer may also be prepared bypolymerization of a protected-thiol monomer. After polymerization, thethiol groups may then be deprotected to give free thiol groups which maythen be reacted under oxidation conditions to form a disulfidelinkage(s). Suitable oxidation conditions include, for example, airoxidation and the use of an oxidizing reagent (e.g. ALDRITHIOLcommercially available from Aldrich Chemical Company, Inc., Milwaukee,Wis.).

The degree of association, as described above, of the polymer of thecomposite forming the multi-dimensional polymer network may vary frompartial association to complete association. By varying the degree ofassociation of the polymer, a short chain polymer may be made to exhibitthe characteristics of a long chain polymer while retaining the desiredcharacteristics of a short chain polymer upon disassociation. Forexample, long chain polymer character promotes overall stability, i.e.resistance to degradation, until the target cell is reached while shortchain polymer character promotes DNA release within the target cell.This duality affords a supramolecular complex containing at least onetherapeutic agent and a multi-dimensional polymer network that exhibitsgreater stability in both nonphysiological and physiological conditionsand greater shelf-life stability. Varying the degree of association ofthe polymer of the supramolecular complex also permits controlledrelease of the therapeutic agent.

In a preferred embodiment of the invention, the polymer of the compositeis a substantially linear polymer. A substantially linear polymer may beany suitable substantially linear polymer or substantially linearcopolymer known in the art capable as part of a composite ofassociating, preferably crosslinking, to form a multi-dimensionalpolymer network, as described above. According to the invention, asubstantially linear polymer may be prepared by any means known in theart. Preferably, a substantially linear polymer may be prepared by anysuitable polymerization technique known in the art including, but notlimited to, those described in Trubetskoy et al., Nucleic AcidsResearch, Vol. 26, No. 18, pp 4178-4185 (1998) (e.g. templatepolymerization, step polymerization, chain polymerization). Asubstantially linear polymer may be prepared from a suitable monomer.Examples of suitable monomers for polymerization to form a substantiallylinear polymer include monomers such as, for example,bis(2-aminoethyl)-1,3-propanediamine (AEPD), andN₂,N₂,N₃,N₃-(3′-PEG₅₀₀₀-aminopropane)-bis(2-aminoethyl)-1,3-propanediammoniumdi-trifluoroacetate (AEPD-PEG). The substantially linear polymer mayfurther contain or may be further modified to contain a functional group(e.g. thiol group), as described above. Preferably, the substantiallylinear polymer is linear polyethyleneimine (PEI) or a linearcyclodextrin-containing polymer, more preferably, a linearcyclodextrin-containing polymer. A linear cyclodextrin-containingpolymer may be any water-soluble linear polymer containing at least onecyclodextrin moiety as part of the polymer backbone. More preferably,the linear cyclodextrin-containing polymer is a linear cyclodextrincopolymer or a linear oxidized cyclodextrin copolymer, each as describedbelow.

A linear cyclodextrin copolymer is a polymer containing cyclodextrinmoieties as an integral part of its polymer backbone. Previously,cyclodextrin moieties were not a part of the main polymer chain butrather attached off a polymer backbone as pendant moieties.

A linear cyclodextrin copolymer has a repeating unit of formula Ia, Ib,or a combination thereof:

In formulae Ia and Ib, C is a substituted or unsubstituted cyclodextrinmonomer and A is a comonomer bound, i.e. covalently bound, tocyclodextrin C. Polymerization of a cyclodextrin monomer C precursorwith a comonomer A precursor results in a linear cyclodextrin copolymer.Within a single linear cyclodextrin copolymer, the cyclodextrin monomerC unit may be the same or different and, likewise, the comonomer A maybe the same or different.

A cyclodextrin monomer precursor may be any cyclodextrin or derivativethereof known in the art. As discussed above, a cyclodextrin is definedas a cyclic polysaccharide most commonly containing six to eightnaturally occurring D(+)-glucopyranose units in an α-(1,4) linkage.Preferably, the cyclodextrin monomer precursor is a cyclodextrin havingsix, seven and eight glucose units, i.e., respectively, an alpha(α)-cyclodextrin, a beta (β)-cyclodextrin and a gamma (γ)-cyclodextrin.A cyclodextrin derivative may be any substituted cyclodextrin known inthe art where the substituent does not interfere with copolymerizationwith comonomer A precursor as described below. A cyclodextrin derivativemay be neutral, cationic or anionic. Examples of suitable substituentsinclude, but are not limited to, hydroxyalkyl groups, such as, forexample, hydroxypropyl, hydroxyethyl; ether groups, such as, forexample, dihydroxypropyl ethers, methyl-hydroxyethyl ethers,ethyl-hydroxyethyl ethers, and ethyl-hydroxypropyl ethers; alkyl groups,such as, for example, methyl; saccharides, such as, for example,glucosyl and maltosyl; acid groups, such as, for example, carboxylicacids, phosphorous acids, phosphinous acids, phosphonic acids,phosphoric acids, thiophosphonic acids, and sulfonic acids; imidazolegroups; sulfate groups; and protected thiol groups.

A cyclodextrin monomer precursor may be further chemically modified(e.g. halogenated, aminated) to facilitate or affect copolymerization ofthe cyclodextrin monomer precursor with a comonomer A precursor, asdescribed below. Chemical modification of a cyclodextrin monomerprecursor allows for polymerization at only two positions on eachcyclodextrin moiety, i.e. the creation of a bifunctional cyclodextrinmoiety. The numbering scheme for the C1-C6 positions of eachglucopyranose ring is as follows:

In a preferred embodiment, polymerization occurs at two of any C2, C3and C6 position, including combinations thereof, of the cyclodextrinmoiety. For example, one cyclodextrin monomer precursor may bepolymerized at two C6 positions while another cyclodextrin monomerprecursor may be polymerized at a C2 and a C6 position of thecyclodextrin moiety. Using β-cyclodextrin as an example, the letteringscheme for the relative position of each glucopyranose ring in acyclodextrin is as follows:

In a preferred embodiment of a linear cyclodextrin copolymer, thecyclodextrin monomer C has the following general formula (II):

In formula (II), n and m represent integers which, along with the othertwo glucopyranose rings, define the total number of glucopyranose unitsin the cyclodextrin monomer. Formula (II) represents a cyclodextrinmonomer which is capable of being polymerized at two C6 positions on thecyclodextrin unit. Examples of cyclodextrin monomers of formula (II)include, but are not limited to, 6^(A)′6^(B)-dideoxy-α-cyclodextrin(n=0, m=4), 6^(A),6^(C)-dideoxy-α-cyclodextrin (n=1, m=3),6^(A),6^(D)-dideoxy-α-cyclodextrin (n=2, m=2),6^(A),6^(B)-dideoxy-β-cyclodextrin (n=0, m=5),6^(A),6^(C)-dideoxy-β-cyclodextrin (n=l, m=4),6^(A),6^(D)-dideoxy-β-cyclodextrin (n=2, m=3),6^(A),6^(B)-dideoxy-γ-cyclodextrin (n=0, m=6),6^(A),6^(C)-dideoxy-γ-cyclodextrin (n=1, m=5),6^(A),6^(D)-dideoxy-γ-cyclodextrin (n=2, m=4), and6^(A),6^(E)-dideoxy-γ-cyclodextrin (n=3, m=3). In another preferredembodiment of a linear cyclodextrin copolymer, a cyclodextrin monomer Cunit has the following general formula (III):

where p=5-7. In formula (III), at least one of D(+)-glucopyranose unitsof a cyclodextrin monomer has undergone ring opening to allow forpolymerization at a C2 and a C3 position of the cyclodextrin unit.Cyclodextrin monomers of formula (III) such as, for example,2^(A),3^(A)-diamino-2^(A),3^(A)-dideoxy-β-cyclodextrin and2^(A),3^(A)-dialdehyde-2^(A),3^(A)-dideoxy-β-cyclodextrin arecommercially available from Carbomer of Westborough, Mass. Examples ofcyclodextrin monomers of formula (III) include, but are not limited to,2^(A),3^(A)-dideoxy-2^(A),3^(A)-dihydro α-cyclodextrin,2^(A),3^(A)-dideoxy-2^(A),3^(A)-dihydro-β-cyclodextrin,2^(A),3^(A)-dideoxy-2^(A),3^(A)-dihydro-γ-cyclodextrin, commonlyreferred to as, respectively, 2,3-dideoxy-α-cyclodextrin,2,3-dideoxy-β-cyclodextrin, and 2,3-dideoxy-γ-cyclodextrin.

A comonomer A precursor may be any straight chain or branched, symmetricor asymmetric compound which upon reaction with a cyclodextrin monomerprecursor, as described above, links two cyclodextrin monomers together.Preferably, a comonomer A precursor is a compound containing at leasttwo functional groups through which reaction and thus linkage of thecyclodextrin monomers can be achieved. Examples of possible functionalgroups, which may be the same or different, terminal or internal, ofeach comonomer A precursor include, but are not limited to, amino, acid,ester, imidazole, and acyl halide groups and derivatives thereof. In apreferred embodiment, the two functional groups are the same andterminal. Upon copolymerization of a comonomer A precursor with acyclodextrin monomer precursor, two cyclodextrin monomers may be linkedtogether by joining the primary hydroxyl side of one cyclodextrinmonomer with the primary hydroxyl side of another cyclodextrin monomer,by joining the secondary hydroxyl side of one cyclodextrin monomer withthe secondary hydroxyl side of another cyclodextrin monomer, or byjoining the primary hydroxyl side of one cyclodextrin monomer with thesecondary hydroxyl side of another cyclodextrin monomer. Accordingly,combinations of such linkages may exist in the final copolymer. Both thecomonomer A precursor and the comonomer A of the final copolymer may beneutral, cationic (e.g. by containing protonated groups such as, forexample, quaternary ammonium groups) or anionic (e.g. by containingdeprotonated groups, such as, for example, sulfate, phosphate orcarboxylate anionic groups). The counterion of a charged comonomer Aprecursor or comonomer A may be any suitable counteranion orcountercation (e.g. the counteranion of a cationic comonomer A precursoror comonomer A may be a halide (e.g. chloride) anion). The charge ofcomonomer A of the copolymer may be adjusted by adjusting pH conditions.Examples of suitable comonomer A precursors include, but are not limitedto, cystamine, 1,6-diaminohexane, diimidazole, dithioimidazole,spermine, dithiospermine, dihistidine, dithiohistidine, succinimide(e.g. dithiobis(succinimidyl propionate) (DSP) and disuccinimidylsuberate (DSS)), and imidates (e.g. dimethyl3,3′-dithiobispropion-imidate (DTBP)). Copolymerization of a comonomer Aprecursor with a cyclodextrin monomer precursor leads to the formationof a linear cyclodextrin copolymer containing comonomer A linkages ofthe following general formulae:

In the above formulae, x=1-50, and y+z=x. Preferably, x=1-30. Morepreferably, x=1-20. In a preferred embodiment, comonomer A isbiodegradable or acid-labile. Also in a preferred embodiment, thecomonomer A precursor and hence the comonomer A may be selectivelychosen in order to achieve a desired application. For example, todeliver small molecular therapeutic agents, a charged polymer may not benecessary and the comonomer A may be a polyethylene glycol group.

In a preferred embodiment of the invention, a linear cyclodextrincopolymer may be prepared by copolymerizing a cyclodextrin monomerprecursor disubstituted with an appropriate leaving group with acomonomer A precursor capable of displacing the leaving groups. Theleaving group, which may be the same or different, may be any leavinggroup known in the art which may be displaced upon copolymerization witha comonomer A precursor. In a preferred embodiment, a linearcyclodextrin copolymer may be prepared by iodinating a cyclodextrinmonomer precursor to form a diiodinated cyclodextrin monomer precursorand copolymerizing the diiodinated cyclodextrin monomer precursor with acomonomer A precursor to form a linear cyclodextrin copolymer having arepeating unit of formula Ia, Ib, or a combination thereof, each asdescribed above. In a preferred embodiment, a method of preparing alinear cyclodextrin iodinates a cyclodextrin monomer precursor asdescribed above to form a diiodinated cyclodextrin monomer precursor offormula IVa, IVb, IVc or a mixture thereof:

The diiodinated cyclodextrin may be prepared by any means known in theart (see, e.g., Tabushi et al. J. Am. Chem. 106, 5267-5270 (1984);Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984)). For example,β-cyclodextrin may be reacted with biphenyl-4,4′-disulfonyl chloride inthe presence of anhydrous pyridine to form a biphenyl-4,4′-disulfonylchloride capped β-cyclodextrin which may then be reacted with potassiumiodide to produce diiodo-β-cyclodextrin. The cyclodextrin monomerprecursor is iodinated at only two positions. By copolymerizing thediiodinated cyclodextrin monomer precursor with a comonomer A precursor,as described above, a linear cyclodextrin polymer having a repeatingunit of formula Ia, Ib, or a combination thereof, also as describedabove, may be prepared. If appropriate, the iodine or iodo groups may bereplaced with other known leaving groups.

The iodo groups or other appropriate leaving group may be displaced witha group that permits reaction with a comonomer A precursor, as describedabove. For example, a diiodinated cyclodextrin monomer precursor offormula IVa, IVb, IVc or a mixture thereof may be aminated to form adiaminated cyclodextrin monomer precursor of formula Va, Vb, Vc or amixture thereof:

The diaminated cyclodextrin monomer precursor may be prepared by anymeans known in the art (see, e.g., Tabushi et al. Tetrahedron Lett.18:1527-1530 (1977); Mungall et al., J. Org. Chem. 1659-1662 (1975)).For example, a diiodo-β-cyclodextrin may be reacted with sodium azideand then reduced to form a diamino-β-cyclodextrin. The cyclodextrinmonomer precursor is aminated at only two positions. The diaminatedcyclodextrin monomer precursor may then be copolymerized with acomonomer A precursor, as described above, to produce a linearcyclodextrin copolymer having a repeating unit of formula Ia, Ib, or acombination thereof, also as described above. However, the aminofunctionality of a diaminated cyclodextrin monomer precursor need not bedirectly attached to the cyclodextrin moiety. Alternatively, the aminofunctionality may be introduced by displacement of the iodo or otherappropriate leaving groups of a cyclodextrin monomer precursor withamino group containing moieties such as, for example, ⁻SCH₂CH₂NH₂, toform a diaminated cyclodextrin monomer precursor of formula Vd, Ve, Vfor a mixture thereof:

A linear cyclodextrin copolymer may also be prepared by reducing alinear oxidized cyclodextrin copolymer, as described below. This methodmay be performed as long as the comonomer A does not contain a reduciblemoiety or group such as, for example, a disulfide linkage.

A linear cyclodextrin copolymer may be oxidized so as to introduce atleast one oxidized cyclodextrin monomer into the copolymer such that theoxidized cyclodextrin monomer is an integral part of the polymerbackbone. A linear cyclodextrin copolymer which contains at least oneoxidized cyclodextrin monomer is defined as a linear oxidizedcyclodextrin copolymer. The cyclodextrin monomer may be oxidized oneither the secondary or primary hydroxyl side of the cyclodextrinmoiety. If more than one oxidized cyclodextrin monomer is present in alinear oxidized cyclodextrin copolymer, the same or differentcyclodextrin monomers oxidized on either the primary hydroxyl side, thesecondary hydroxyl side, or both may be present. For illustrationpurposes, a linear oxidized cyclodextrin copolymer with oxidizedsecondary hydroxyl groups has, for example, at least one unit of formulaVIa or VIb:

In formulae VIa and VIb, C is a substituted or unsubstituted oxidizedcyclodextrin monomer and A is a comonomer bound, i.e. covalently bound,to the oxidized cyclodextrin C. Also in formulae VIa and VIb, oxidationof the secondary hydroxyl groups leads to ring opening of thecyclodextrin moiety and the formation of aldehyde groups.

A linear oxidized cyclodextrin copolymer may be prepared by oxidation ofa linear cyclodextrin copolymer as discussed above. Oxidation of alinear cyclodextrin copolymer may be accomplished by oxidationtechniques known in the art. (Hisamatsu et al., Starch 44:188-191(1992)). Preferably, an oxidant such as, for example, sodium periodateis used. It would be understood by one of ordinary skill in the art thatunder standard oxidation conditions that the degree of oxidation mayvary or be varied per copolymer. Thus in one embodiment, a linearoxidized copolymer may contain one oxidized cyclodextrin monomer. Inanother embodiment, substantially all to all cyclodextrin monomers ofthe copolymer would be oxidized.

Another method of preparing a linear oxidized cyclodextrin copolymerinvolves the oxidation of a diiodinated or diaminated cyclodextrinmonomer precursor, as described above, to form an oxidized diiodinatedor diaminated cyclodextrin monomer precursor and copolymerization of theoxidized diiodinated or diaminated cyclodextrin monomer precursor with acomonomer A precursor. In a preferred embodiment, an oxidizeddiiodinated cyclodextrin monomer precursor of formula VIIa, VIIb, VIIc,or a mixture thereof:

may be prepared by oxidation of a diiodinated cyclodextrin monomerprecursor of formulae IVa, IVb, IVc, or a mixture thereof, as describedabove. In another preferred embodiment, an oxidized diaminatedcyclodextrin monomer precursor of formula VIIIa, VIIIb, VIIIc or amixture thereof:

may be prepared by amination of an oxidized diiodinated cyclodextrinmonomer precursor of formulae VIIa, VIIb, VIIc, or a mixture thereof, asdescribed above. In still another preferred embodiment, an oxidizeddiaminated cyclodextrin monomer precursor of formula IXa, IXb, IXc or amixture thereof:

may be prepared by displacement of the iodo or other appropriate leavinggroups of an oxidized cyclodextrin monomer precursor disubstituted withan iodo or other appropriate leaving group with the amino groupcontaining moiety ⁻SCH₂CH₂NH₂.

Alternatively, an oxidized diiodinated or diaminated cyclodextrinmonomer precursor, as described above, may be prepared by oxidizing acyclodextrin monomer precursor to form an oxidized cyclodextrin monomerprecursor and then diiodinating and/or diaminating the oxidizedcyclodextrin monomer, as described above. As discussed above, thecyclodextrin moiety may be modified with other leaving groups other thaniodo groups and other amino group containing functionalities. Theoxidized diiodinated or diaminated cyclodextrin monomer precursor maythen be copolymerized with a comonomer A precursor, as described above,to form a linear oxidized cyclodextrin copolymer.

In a preferred embodiment of the invention, a linear cyclodextrincopolymer or a linear oxidized cyclodextrin copolymer terminates with atleast one comonomer A precursor or hydrolyzed product of the comonomer Aprecursor, each as described above. As a result of termination of thecyclodextrin copolymer with at least one comonomer A precursor, at leastone free functional group, as described above, exists per linearcyclodextrin copolymer or per linear oxidized cyclodextrin copolymer.For example, the functional group may be an acid group or a functionalgroup that may be hydrolyzed to an acid group. According to theinvention, the functional group may be further chemically modified asdesired to enhance the properties of the cyclodextrin copolymer, suchas, for example, colloidal stability and transfection efficiency. Forexample, the functional group may be modified by reaction with PEG toform a PEG terminated cyclodextrin copolymer to enhance colloidalstability or with histidine to form an imidazolyl terminatedcyclodextrin copolymer to enhance intracellular (e.g. endosomal release)and transfection efficiency.

Further chemistry may be performed on the cyclodextrin copolymer throughthe modified functional group. For example, the modified functionalgroup may be used to extend a polymer chain by linking a linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer, asdescribed herein, to the same or different cyclodextrin copolymer or toa non-cyclodextrin polymer. In a preferred embodiment of the invention,the polymer to be added on is the same or different linear cyclodextrincopolymer or linear oxidized cyclodextrin copolymer which may alsoterminate with at least one comonomer A precursor for furthermodification, each as described herein.

Alternatively, at least two of the same or different linear cyclodextrincopolymers or linear oxidized cyclodextrin copolymers containing aterminal functional group or a terminal modified functional group, asdescribed above, may be reacted and linked together through thefunctional or modified functional group. Preferably, upon reaction ofthe functional or modified functional groups, a degradable moiety suchas, for example, a disulfide linkage is formed. For example,modification of the terminal functional group with cysteine may be usedto produce a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer having at least one free thiol group. Reactionwith the same or different cyclodextrin copolymer also containing atleast one free thiol group will form a disulfide linkage between the twocopolymers. In a preferred embodiment of the invention, the functionalor modified functional groups may be selected to offer linkagesexhibiting different rates of degradation (e.g. via enzymaticdegradation) and thereby provide, if desired, a time release system fora therapeutic agent. The resulting polymer may be crosslinked, asdescribed herein. A therapeutic agent, as described herein, may be addedprior to or post crosslinking of the polymer. A ligand, as describedherein, may also be bound through the modified functional group.

According to the invention, a linear cyclodextrin copolymer or linearoxidized cyclodextrin copolymer may be attached to or grafted onto asubstrate. The substrate may be any substrate as recognized by those ofordinary skill in the art. In another preferred embodiment of theinvention, a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer may be crosslinked to a polymer to form,respectively, a crosslinked cyclodextrin copolymer or a crosslinkedoxidized cyclodextrin copolymer. The polymer may be any polymer capableof crosslinking with a linear or linear oxidized cyclodextrin copolymerof the invention (e.g. polyethylene glycol (PEG) polymer, polyethylenepolymer). The polymer may also be the same or different linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer. Thus,for example, a linear cyclodextrin copolymer may be crosslinked to anypolymer including, but not limited to, itself, another linearcyclodextrin copolymer, and a linear oxidized cyclodextrin copolymer. Acrosslinked linear cyclodextrin copolymer of the invention may beprepared by reacting a linear cyclodextrin copolymer with a polymer inthe presence of a crosslinking agent. A crosslinked linear oxidizedcyclodextrin copolymer of the invention may be prepared by reacting alinear oxidized cyclodextrin copolymer with a polymer in the presence ofan appropriate crosslinking agent. The crosslinking agent may be anycrosslinking agent known in the art. Examples of crosslinking agentsinclude dihydrazides and dithiols. In a preferred embodiment, thecrosslinking agent is a labile group such that a crosslinked copolymermay be uncrosslinked if desired.

A linear cyclodextrin copolymer and a linear oxidized cyclodextrincopolymer of the invention may be characterized by any means known inthe art. Such characterization methods or techniques include, but arenot limited to, gel permeation chromatography (GPC), matrix assistedlaser desorption ionization-time of flight mass spectrometry (MALDI-TOFMass spec), ¹H and ¹³C NMR, light scattering and titration.

In another preferred embodiment of the invention, the polymer of thecomposite is a substantially branched polymer such as, for example,branched polyethyleneimine (PEI) or a branched cyclodextrin-containingpolymer, preferably, a branched cyclodextrin-containing polymer. Abranched cyclodextrin-containing polymer may be any water-solublebranched polymer containing at least one cyclodextrin moiety which maybe a part of the polymer backbone and/or pendant from the polymerbackbone. Preferably, a branched cyclodextrin-containing polymer is abranched cyclodextrin copolymer or a branched oxidized cyclodextrincopolymer. A branched cyclodextrin copolymer or a branched oxidizedcyclodextrin copolymer is, respectively, a linear cyclodextrin copolymeror a linear oxidized cyclodextrin copolymer, as described above, fromwhich a subordinate chain is branched. The branching subordinate chainmay be any saturated or unsaturated, linear or branched hydrocarbonchain. The branching subordinate chain may further contain variousfunctional groups or substituents such as, for example, hydroxyl, amino,acid, ester, amido, keto, formyl, and nitro groups. The branchingsubordinate chain may also contain at least one cyclodextrin moiety. Thebranching subordinate chain may also be modified with a ligand, asdescribed herein. Such ligand modification includes, but is not limitedto, attachment of a ligand to a cyclodextrin moiety in the branchingsubordinate chain. Preferably, the branched cyclodextrin-containingpolymer is a branched cyclodextrin copolymer or a branched oxidizedcyclodextrin copolymer, as defined above, of which the branchingsubordinate chain contains at least one cyclodextrin moiety. Accordingto the invention, if the branching subordinate chain contains at leastone cyclodextrin moiety, the cyclodextrin moiety may facilitateencapsulation of a therapeutic agent, each as described herein.Preferably, a cyclodextrin moiety of a branching subordinate chainfacilitates encapsulation of a therapeutic agent in conjunction with acyclodextrin moiety in the polymer backbone. A branchedcyclodextrin-containing polymer may be prepared by any means known inthe art including, but not limited to, derivatization (e.g.substitution) of a polymer (e.g. linear or branched PEI) with acyclodextrin monomer precursor, as defined above. A branchedcyclodextrin-containing polymer of the invention may be characterized byany means known in the art. Such characterization methods or techniquesinclude, but are not limited to, gel permeation chromatography (GPC),matrix assisted laser desorption ionization-time of flight massspectrometry (MALDI-TOF Mass spec), ¹H and ¹³C NMR, light scattering andtitration.

According to the invention, a branched cyclodextrin-containing polymermay be crosslinked under crosslinking reaction conditions, each asdescribed above. In a preferred embodiment of the invention, a branchedcyclodextrin-containing polymer is crosslinked with itself. In anotherpreferred embodiment of the invention, a branchedcyclodextrin-containing polymer is crosslinked with a polymer. Thepolymer may be the same or different branched cyclodextrin-containingpolymer, a substantially linear polymer, or a substantially branchedpolymer, each as described above.

According to the invention, a substantially branched polymer may beattached to or grafted onto a substrate, as described above. Furtherchemistry may be performed on the substantially branched polymer througha modified functional group, as described above.

A poly(ethylenimine) (PEI) for use in the invention has a weight averagemolecular weight of between about 800 and about 800,000 daltons,preferably, between about 2,000 and 100,000 daltons, more preferably,between about 2,000 and about 25,000 daltons. The PEI may be linear orbranched. Suitable PEI compounds are commercially available from manysources, including polyethylenimine from Aldrich Chemical Company,polyethylenimine from Polysciences, and POLYMIN poly(ethylenimine) andLUPASOL™ poly(ethylenimine) available from BASF Corporation.

According to the invention, a polymer of the composite, or one of themonomers which form a polymer of the composite, may be modified with atleast one ligand such that the resulting composite or supramolecularcomplex is associated with at least one ligand, each as describedherein. Alternatively, according to a method of the invention, once acomposite or a supramolecular complex is formed, it may then becontacted with a ligand such that the composite or supramolecularcomplex is modified with at least one ligand in such a way that theligand is associated with the composite or supramolecular complex, eachas described herein. The ligand of such a ligand-containing composite orligand-containing supramolecular complex allows for targeting and/orbinding to a desired cell. If more than one ligand is attached, theligand may be the same or different. Examples of suitable ligandsinclude, but are not limited to, vitamins (e.g. folic acid), proteins(e.g. transferrin, and monoclonal antibodies) and polysaccharides. Thechoice of ligand may vary depending upon the type of delivery desired.For example, receptor-mediated delivery may by achieved by, but notlimited to, the use of a folic acid ligand while antisense oligodelivery may be achieved by, but not limited to, use of a transferrinligand.

The ligand may be associated with the composite or supramolecularcomplex by means known in the art. For example, a linear cyclodextrincopolymer or linear oxidized cyclodextrin copolymer may be modified withat least one ligand attached to the cyclodextrin copolymer. The ligandmay be attached to the cyclodextrin copolymer through the cyclodextrinmonomer C or comonomer A. Preferably, the ligand is attached to at leastone cyclodextrin moiety of the cyclodextrin copolymer. Preferably, theligand allows a cyclodextrin copolymer to target and bind to a cell. Ifmore than one ligand, which may be the same or different, is attached toa cyclodextrin copolymer, the additional ligand or ligands may be boundto the same or different cyclodextrin moiety or the same or differentcomonomer A of the copolymer. A cyclodextrin copolymer may also befurther modified to contain a functional group to promote association ofthe cyclodextrin copolymer with the therapeutic agent and/or otherpolymer(s) of the composite.

According to a method of the invention, upon formation of thesupramolecular complex, the therapeutic agent becomes encapsulated inthe multi-dimensional polymer network created from the polymer of acomposite, as described above. Encapsulation is defined as any means bywhich, the therapeutic agent associates (e.g. electrostatic interaction,hydrophobic interaction, actual encapsulation) with themulti-dimensional polymer network. The degree of association may bedetermined by techniques known in the art including, for example,fluorescence studies, DNA mobility studies, light scattering, electronmicroscopy, and will vary depending upon the therapeutic agent. As amode of delivery, for example, a supramolecular complex containing amulti-dimensional polymer network created from the polymer of acomposite, as described above, and DNA may be used to aid intransfection, i.e. the uptake of DNA into an animal (e.g. human) cell.(Boussif, O. Proceedings of the National Academy of Sciences,92:7297-7301 (1995); Zanta et al. Bioconjugate Chemistry, 8:839-844(1997)).

The therapeutic agent is not an integral part of the multi-dimensionalpolymer network of the supramolecular complex. Upon encapsulation, thetherapeutic agent may or may not retain its biological or therapeuticactivity. Regardless, upon decomplexation or uncrosslinking of thesupramolecular complex, specifically, of the multi-dimensional polymernetwork, the activity of the therapeutic agent is restored. Accordingly,encapsulation of the therapeutic agent affords, advantageously,protection against loss of activity due to, for example, degradation andoffers enhanced bioavailability. Encapsulation of a lipophilictherapeutic agent offers enhanced, if not complete, solubility of thelipophilic therapeutic agent. The therapeutic agent may be furthermodified with at least one ligand prior to or after composite orsupramolecular complex formation, as described above.

The therapeutic agent may be any lipophilic or hydrophilic, synthetic ornaturally occurring biologically active therapeutic agent includingthose known in the art. Examples of suitable therapeutic agents include,but are not limited to, antibiotics, steroids, polynucleotides (e.g.genomic DNA, cDNA, mRNA and antisense oligonucleotides), plasmids,peptides, peptide fragments, small molecules (e.g. doxorubicin),chelating agents (e.g. deferoxamine (DESFERAL),ethylenediaminetetraacetic acid (EDTA)), natural products (e.g. Taxol,Amphotericin), and other biologically active macromolecules such as, forexample, proteins and enzymes.

A supramolecular complex of the invention may be, for example, a solid,liquid, suspension, or emulsion. Preferably a supramolecular complex ofthe invention is in a form that can be injected intravenously. Othermodes of administration of a supramolecular complex of the inventioninclude, depending on the state of the supramolecular complex, methodsknown in the art such as, but not limited to, oral administration,topical application, parenteral, intravenous, intranasal, intraocular,intracranial or intraperitoneal injection. Prior to administration, asupramolecular complex may be isolated and purified by any means knownin the art including, for example, centrifugation, dialysis and/orlyophilization.

Depending upon the type of therapeutic agent used, a supramolecularcomplex of the invention may be used in a variety of therapeutic methods(e.g. DNA vaccines, antibiotics, antiviral agents) for the treatment ofinherited or acquired disorders such as, for example, cystic fibrosis,Gaucher's disease, muscular dystrophy, AIDS, cancers (e.g., multiplemyeloma, leukemia, melanoma, and ovarian carcinoma), cardiovascularconditions (e.g., progressive heart failure, restenosis, andhemophilia), and neurological conditions (e.g., brain trauma). Accordingto the invention, a method of treatment administers a therapeuticallyeffective amount of a supramolecular complex as prepared by a method ofthe invention. A therapeutically effective amount, as recognized bythose of skill in the art, will be determined on a case by case basis.Factors to be considered include, but are not limited to, the disorderto be treated and the physical characteristics of the one suffering fromthe disorder.

In another embodiment of the invention, the therapeutic agent is atleast one biologically active compound having agricultural utility. Theagriculturally biologically active compounds include those known in theart. For example, suitable agriculturally biologically active compoundsinclude, but are not limited to, fungicides, herbicides, insecticides,and mildewcides.

The following examples are given to illustrate the invention. It shouldbe understood, however, that the invention is not to be limited to thespecific conditions or details described in these examples.

EXAMPLES Materials

β-cyclodextrin (Cerestar USA, Inc. of Hammond, Ind.) was dried in vacuo(<0.1 mTorr) at 120° C. for 12 h before use. Biphenyl-4,4′-disulfonylchloride (Aldrich Chemical Company, Inc. of Milwaukee, Wis.) wasrecrystallized from chloroform/hexanes. Potassium iodide was powderedwith a mortar and pestle and dried in an oven at 200° C. All otherreagents were obtained from commercial suppliers and were used asreceived without further purification. Polymer samples were analyzed ona Hitachi HPLC system equipped with an Anspec R1 detector, a PrecisionDetectors DLS detector, and a Progel-TSK G3000_(PWXL) column using 0.3 MNaCl or water as eluant at a 1.0 mLmin⁻¹ flow rate.

Example 1 Biphenyl-4,4′-disulfonyl-A,D-Capped β-Cyclodextrin, 1 (Tabushiet al. J. Am. Chem. Soc. 106, 5267-5270 (1984))

A 500 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 7.92 g (6.98 mmol) of dryβ-cyclodextrin and 250 mL of anhydrous pyridine (Aldrich ChemicalCompany, Inc.). The resulting solution was stirred at 50° C. undernitrogen while 2.204 g (6.28 mmol) of biphenyl-4,4′-disulfonyl chloridewas added in four equal portions at 15 min intervals. After stirring at50° C. for an additional 3 h, the solvent was removed in vacuo and theresidue was subjected to reversed-phase column chromatography using agradient elution of 0-40% acetonitrile in water. Fractions were analyzedby high performance liquid chromatography (HPLC) and the appropriatefractions were combined. After removing the bulk of the acetonitrile ona rotary evaporator, the resulting aqueous suspension was lyophilized todryness. This afforded 3.39 g (38%) of 1 as a colorless solid.

Example 2 6^(A),6^(D)-Diiodo-6^(A),6^(D)-Dideoxy-β-cyclodextrin, 2(Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))

A 40 mL centrifuge tube equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.02 g (7.2 mmol) of 1, 3.54 g(21.3 mmol) of dry, powdered potassium iodide (Aldrich) and 15 mL ofanhydrous N,N-dimethylformamide (DMF) (Aldrich). The resultingsuspension was stirred at 80° C. under nitrogen for 2 h. After coolingto room temperature, the solids were separated by filtration and thesupernatant was collected. The solid precipitate was washed with asecond portion of anhydrous DMF and the supernatants were combined andconcentrated in vacuo. The residue was then dissolved in 14 mL of waterand cooled in an ice bath before 0.75 mL (7.3 mmol) oftetrachloroethylene (Aldrich) was added with rapid stirring. Theprecipitated inclusion complex was filtered on a medium glass frit andwashed with a small portion of acetone before it was dried under vacuumover P₂O₅ for 14 h. This afforded 0.90 g (92%) of 2 as a white solid.

Example 3 6^(A),6^(D)-Diazido-6^(A),6^(D)-Dideoxy-β-cyclodextrin, 3(Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))

A 100 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.704 g (1.25 mmol) ofβ-cyclodextrin diiodide, 0.49 g (7.53 mmol) of sodium azide (EM Scienceof Gibbstown, N.J.) and 10 mL of anhydrous N,N-dimethylformamide (DMF).The resulting suspension was stirred at 60° C. under nitrogen for 14 h.The solvent was then removed in vacuo. The resulting residue wasdissolved in enough water to make a 0.2 M solution in salt and thenpassed through 11.3 g of Biorad AG501-X8(D) resin to remove residualsalts. The eluant was then lyophilized to dryness yielding 1.232 g (83%)of 3 as a white amorphous solid which was carried on to the next stepwithout further purification.

Example 4 6^(A),6^(D)-Diamino-6^(A),6^(D)-Dideoxy-β-cyclodextrin, 4(Mungall et al., J. Org. Chem. 1659-1662 (1975))

A 250 mL round bottom flask equipped with a magnetic stirbar and aseptum was charged with 1.232 g (1.04 mmol) of β-cyclodextrin bisazideand 50 mL of anhydrous pyridine (Aldrich). To this stirring suspensionwas added 0.898 g (3.42 mmol) of triphenylphosphine. The resultingsuspension was stirred for 1 h at ambient temperature before 10 mL ofconcentrated aqueous ammonia was added. The addition of ammonia wasaccompanied by a rapid gas evolution and the solution becamehomogeneous. After 14 h, the solvent was removed in vacuo and theresidue was triturated with 50 mL of water. The solids were filtered offand the filtrate was made acidic (pH<4) with 10% HCl before it wasapplied to an ion exchange column containing Toyopearl SP-650M (NH₄ ⁺form) resin. The product 4 was eluted with a gradient of 0-0.5 Mammonium bicarbonate. Appropriate fractions were combined andlyophilized to yield 0.832 g (71%) of the product 4 as the bis(hydrogencarbonate) salt.

Example 5 β-Cyclodextrin-DSP copolymer, 5

A 20 mL scintillation vial was charged with a solution of 92.6 mg(7.65×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before asolution of 30.9 mg (7.65×10⁻⁵ mol) of dithiobis(succinimidylpropionate) (DSP, Pierce Chemical Co. of Rockford, Ill.) in 1 mL ofchloroform was added. The resulting biphasic mixture was agitated with aVortex mixer for 0.5 h. The aqueous layer was then decanted andextracted with 3×1 mL of fresh chloroform. The aqueous polymer solutionwas then subjected to gel permeation chromatography (GPC) on ToyopearlHW-40F resin using water as eluant. Fractions were analyzed by GPC andappropriate fractions were lyophilized to yield 85 mg (85%) as acolorless amorphous powder.

Example 6 β-cyclodextrin-DSS copolymer, 6

A β-cyclodextrin-DSS copolymer, 6, was synthesized in a manner analogousto the DSP polymer, 5, except that disuccinimidyl suberate (DSS, PierceChemical Co. of Rockford, Ill.) was substituted for the DSP reagent.Compound 6 was obtained in 67% yield.

Example 7 β-Cyclodextrin-DTBP copolymer, 7

A 20 mL scintillation vial was charged with a solution of 91.2 mg(7.26×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before22.4 mg (7.26×10⁻⁵ mol) of dimethyl 3,3′-dithiobis(propionimidate)₂ HCl(DTBP, Pierce Chemical Co of Rockford, Ill.) was added. The resultinghomogeneous solution was agitated with a Vortex mixer for 0.5 h. Theaqueous polymer solution was then subjected to gel permeationchromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzedby GPC and appropriate fractions were lyophilized to yield 67 mg (67%)of a colorless amorphous powder.

Example 8 Polyethylene glycol 600 dihydrazide, 8

A 100 mL round bottom flask equipped with a magnetic stirbar and areflux condenser was charged with 1.82 g (3.0 mmol) of polyethyleneglycol 600 (Fluka Chemical Corp of Milwaukee, Wis.), 40 mL of absoluteethanol (Quantum Chemicals Pty Ltd of Tuscola, Ill.) and a few drops ofsulfuric acid. The resulting solution was heated to reflux for 14 h.Solid sodium carbonate was added to quench the reaction and the solutionof the PEG diester was transferred under nitrogen to an addition funnel.This solution was then added dropwise to a solution of 0.6 mL (9.0 mmol)of hydrazine hydrate (Aldrich) in 10 mL of absolute ethanol. A smallamount of a cloudy precipitate formed. The resulting solution was heatedto reflux for 1 h before it was filtered and concentrated. GPC analysisrevealed a higher molecular weight impurity contaminating the product.Gel permeation chromatography on Toyopearl HW-40 resin enabled a partialpurification of this material to approximately 85% purity.

Example 9 Oxidation of β-cyclodextrin-DSS copolymer, 9 (Hisamatsu etal., Starch 44, 188-191 (1992))

The β-cyclodextrin-DSS copolymer 6 (92.8 mg, 7.3×10⁻⁵ mol) was dissolvedin 1.0 mL of water and cooled in an ice bath before 14.8 mg (7.3×10⁻⁵mol) of sodium periodate was added. The solution immediately turnedbright yellow and was allowed to stir in the dark at 0° C. for 14 h. Thesolution was then subjected to gel permeation chromatography (GPC) onToyopearl HW-40 resin using water as eluant. Fractions were analyzed byGPC. Appropriate fractions were combined and lyophilized to dryness toyield 84.2 mg (91%) of a light brown amorphous solid.

Example 10 Polyethylene glycol (PEG) 600 diacid chloride, 10

A 50 mL round bottom flask equipped with a magnetic stirbar and a refluxcondenser was charged with 5.07 g (ca. 8.4 mmol) of polyethylene glycol600 diacid (Fluka Chemical Corp of Milwaukee, Wis.) and 10 mL ofanhydrous chloroform (Aldrich). To this stirring solution was added 3.9mL (53.4 mmol) of thionyl chloride (Aldrich) and the resulting solutionwas heated to reflux for 1 h, during which time gas evolution wasevident. The resulting solution was allowed to cool to room temperaturebefore the solvent and excess thionyl chloride were removed in vacuo.The resulting oil was stored in a dry box and used without purification.

Example 11 β-Cyclodextrin-PEG 600 copolymer, 11

A 20 mL scintillation vial was charged with a solution of 112.5 mg(8.95×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of6^(A),6^(D)-diamino-6^(A),6^(D)-dideoxy-β-cyclodextrin, 50 μL (3.6×10⁻⁴mol) of triethylamine (Aldrich), and 5 mL of anhydrousN,N-dimethylacetamide (DMAc, Aldrich). The resulting suspension was thentreated with 58 mg (9.1×10⁻⁵ mol) of polyethylene glycol 600 diacidchloride, 10. The resulting solution was agitated with a Vortex mixerfor 5 minutes and then allowed to stand at 25° C. for 1 h during whichtime it became homogeneous. The solvent was removed in vacuo and theresidue was subjected to gel permeation chromatography on ToyopearlHW-40F resin using water as eluant. Fractions were analyzed by GPC andappropriate fractions were lyophilized to dryness to yield 115 mg (75%)of a colorless amorphous powder.

Example 12 β-Cyclodextrin-DSP copolymer, 12

A 8 mL vial was charged with a solution of 102.3 mg (8.80×10⁻⁵ mol) of2^(A),3^(A)-diamino-2^(A),3^(A)-dideoxy-β-cyclodextrin in 1 mL of water.The pH of the solution was adjusted to 10 with 1 M NaOH before asolution of 36.4 mg (8.80×10⁻⁵ mol) of dithiobis(succinimidylpropionate) (DSP, Pierce Chemical Co. of Rockford, Ill.) in 1 mL ofchloroform was added. The resulting biphasic mixture was agitated with aVortex mixer for 0.5 h. The aqueous layer was then decanted andextracted with 3×1 mL of fresh chloroform. The aqueous polymer solutionwas then subjected to gel permeation chromatography.

Example 136^(A),6^(D)-Bis-(2-aminoethylthio)-6^(A),6^(D)-dideoxy-β-cyclodextrin,13 (Tabushi, I: Shimokawa, K; Fugita, K. Tetrahedron Lett. 1977,1527-1530)

A 25 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 0.91 mL (7.37 mmol) of a 0.81 M solution of sodium2-aminoethylthiolate in ethanol. (Fieser, L. F.; Fieser, M. Reagents forOrganic Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266). Thesolution was evaporated to dryness and the solid was redissolved in 5 mLof anhydrous DMF (Aldrich).6^(A),6^(D)-Diiodo-6^(A),6^(D)-dideoxy-β-cyclodextrin (100 mg, 7.38×10⁻⁵mol) was added and the resulting suspension was stirred at 60° C. undernitrogen for 2 h. After cooling to room temperature, the solution wasconcentrated in vacuo and the residue was redissolved in water. Afteracidifying with 0.1 N HCl, the solution was applied to a ToyopearlSP-650M ion-exchange column (NH₄′ form) and the product was eluted witha 0 to 0.4 M ammonium bicarbonate gradient. Appropriate fractions werecombined and lyophilized to dryness. This afforded 80 mg (79%) of 13 asa white powder.

Example 14 β-Cyclodextrin(cystamine)-DTBP copolymer, 14

A 4 mL vial was charged with a solution of 19.6 mg (1.42×10⁻⁵ mol) ofthe bis(hydrogen carbonate) salt of 13 in 0.5 mL of 0.1 M NaHCO₃. Thesolution was cooled in an ice bath before 4.4 mg (1.4×10⁻⁵ mol) ofdimethyl 3,3′-dithiobispropionimidate-2HCl (DTBP, Pierce Chemical Co. ofRockford, Ill.) was added. The resulting solution was then agitated witha Vortex mixer and allowed to stand at 0° C. for 1 h. The reaction wasquenched with 1M Tris-HCl before it was acidified to pH 4 with 0.1N HCl.The aqueous polymer solution was then subjected to gel permeationchromatography on Toyopearl HW-40F resin. Fractions were analyzed by GPCand appropriate fractions were lyophilized to dryness. This afforded21.3 mg (100%) of 14 as a white powder.

Example 15 β-Cyclodextrin(cystamine)-DMS copolymer, 15

A 10 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 200 mg (1.60×10⁻⁴ mol) of 13, 44 μL (3.2×10⁻⁴ mol) oftriethylamine (Aldrich Chemical Co., Milwaukee, Wis.), 43.6 mg(1.60×10⁻⁴ mol) of dimethylsuberimidate.2HCl(DMS, Pierce Chemical Co. ofRockford, Ill.), and 3 mL of anhydrous DMF (Aldrich Chemical Co.,Milwaukee, Wis.). The resulting slurry was heated to 80° C. for 18 hoursunder a steady stream of nitrogen during which time most of the solventhad evaporated. The residue which remained was redissolved in 10 mL ofwater and the resulting solution was then acidified with 10% HCl to pH4. This solution was then passed through an Amicon Centricon Plus-205,000 NMWL centrifugal filter. After washing with 2×10 mL portions ofwater, the polymer solution was lyophilized to dryness yielding 41.4 mg(18%) of an off-white amorphous solid.

Example 16 Fixed Permanent Charged Copolymer Complexation with Plasmid

In general, equal volumes of fixed charged CD-polymer and DNA plasmidsolutions in water are mixed at appropriate polymer/plasmid chargeratios. The mixture is then allowed to equilibrate and self-assemble atroom temperature overnight. Complexation success is monitored bytransferring a small aliquot of the mixture to 0.6% agarose gel andchecking for DNA mobility. Free DNA travels under an applied voltage,whereas complexed DNA is retarded at the well.

1 μg of DNA at a concentration of 0.1 μg/μL in distilled water was mixedwith 10 μL of copolymer 14 at polymer amine: DNA phosphate charge ratiosof 2.4, 6, 12, 24, 36, 60, and 120. The solution was mixed manually by amicropipette and then gently mixed overnight on a lab rotator. 1 μg/μLof loading buffer (40% sucrose, 0.25% bromophenol blue, and 200 mMTris-Acetate buffer containing 5 mM EDTA (Gao et al., Biochemistry35:1027-1036 (1996)) was added to each solution the following morning.Each DNA/polymer sample was loaded on a 0.6% agarose electrophoresis gelcontaining 6 μg of EtBr/100 mL in 1×TAE buffer (40 mM Tris-acetate/1 mMEDTA) and 40V was applied to the gel for 1 hour. The extent ofDNA/polymer complexation was indicated by DNA retardation in the gelmigration pattern. The polymer (14) retarded DNA at charge ratios of 6and above, indicating complexation under these conditions.

Example 17 Crosslinking of Copolymer-Plasmid Complex

Copolymer 14 or copolymer 15 was oxidized as in Example 9. Oxidizedcopolymer 14 or 15 was then complexed with a DNA plasmid as in Example16. The solution was buffered with a borate buffer to pH 8.5 and acrosslinking agent, PEG₆₀₀-Dihydrazide, was then added and thesupramolecular complex formed was analyzed by light scattering, zetapotential, and electron microscopy. Using oxidized copolymer 15, thepolymer-plasmid DNA composite gave an average particle size of 90 nm bylight scattering and had a surface charge of 40 mV as determined by zetapotential measurement. Upon addition of PEG₆₀₀-Dihydrazide, thesupramolecular complex had an average size of 120 nm and a surfacecharge of 17 mV. Electron microscopy showed the composite to be uniformin size while the supramolecular complex revealed some dispersion insize.

Example 18 Transfection Studies with Plasmids Encoding LuciferaseReporter Gene

BHK-21 cells were plated in 24 well plates at a cell density of 60,000cells/well 24 hours before transfection. Plasmids encoding theluciferase gene were mixed with the CD-polymer as in Example 16 exceptcopolymer 14 was replaced with copolymer 15. Media solution containingthe DNA/polymer complexes was added to cultured cells and replaced withfresh media after 24 hours of incubation at 37° C. The cells were lysed48 hours after transfection. Appropriate substrates for the luciferaselight assay were added to the cell lysate. Luciferase activity, measuredin terms of light units produced, was quantified by a luminometer.DNA/polymer complexes successfully transfected BHK-21 cells at a chargeratios of 10, 20, 30, and 40 with maximum transfection at polymeramine:DNA phosphate charge ratio of 40. Cell lysate was also used todetermine cell viability by the Lowry protein assay. (Lowry et al.,Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). No toxicitywas observed up to charge ratios of 40.

Example 19 Transfection Studies with a Supramolecular Complex

The supramolecular complex formed in Example 17 was used to transfectBHK-21 cells following the procedure of Example 18. No transfection wasobserved.

Example 20 Crosslinking of Polyethyleneimine-Plasmid Complex

Polyethyleneimine (PEI) is complexed with a DNA plasmid as in Example16. A crosslinking agent (for example, dimethyl3,3′-dithiobispropionimidate (DTBP, commercially available from PierceChemical Co. of Rockford, Ill.); dithiobis(succinimidyl propionate)(DSP, commercially available from Pierce Chemical Co. of Rockford, Ill.)for biodegradable crosslinking; and disuccinimidyl suberate (DSS,commercially available from Pierce Chemical Co.) or dimethylsuberimidate(DMS, commercially available from Pierce Chemical Co.) for lessbiodegradable crosslinking) is then added and the supramolecular complexformed is analyzed by light scattering, zeta potential, and electronmicroscopy.

Example 21 Crosslinking Polymers Formed From DNA Template Polymerization

Template polymerization using DNA as the template is accomplished asdescribed by Trubetskoy et al. Nucleic Acids Research, Vol. 26, No. 18,pp 4178-4185 (1998).

DNA is contacted with AEPD and comonomers A. The resultant composite ofsubstantially linear polymer and DNA is crosslinked by adding suitablecrosslinking agents (for example, DTBP, DSP, DSS, DMS) and thesupramolecular complex formed is analyzed by light scattering, zetapotential, and electron microscopy.

Example 22 Crosslinking Polymers Formed From DNA Template Polymerization

Template polymerization using DNA as the template is accomplished asdescribed by Trubetskoy et al. Nucleic Acids Research, Vol. 26, No. 18,pp 4178-4185 (1998).

DNA is contacted with oxidized cyclodextrin diamines (for example, IXa,IXb, IXc) and comonomers A. The resultant composite of substantiallylinear polymer and DNA is crosslinked by adding suitable crosslinkingagents (for example, adipic acid dihydrazide, polyethylene glycol 600dihydrazide 8 of Example 8) and the supramolecular complex formed isanalyzed by light scattering, zeta potential, and electron microscopy.

Example 23 Thiolation of Cyclodextrin (CD) Polymer with Traut's Reagent

Under nitrogen, 10.1 mg (7.34×10⁻⁵ mol) of Traut's reagent (PierceChemical Co. of Rockford, Ill.) was added to 1.00 mL of a 5.0 mMsolution of β-CD(cystamine)-DMS copolymer 15 in 0.1 M Na₂CO₃ (pH 10.0)containing 1.0 mM EDTA. The resulting solution was allowed to standunder nitrogen, N2, at ambient temperature for 2 hours. The solution wasthen opened to air and filtered through an Amicon 5,000 NMWL centrifugalfilter after which the supernatant was diluted with 10.0 mL of water andfiltered a second time. The supernatant solution was then diluted to a1.00 mL volume in water and stored under nitrogen. An aliquot wastitrated with Ellman's reagent (Hermanson, G. T., BioconjugateTechniques; Academic: New York, p. 89 (1996)) to yield a thiol contentof 1.56×10⁻⁶ mol, corresponding to thiol functionalization of 31% of thepolymer cyclodextrin moieties.

Example 24 Air Oxidation of Thiolated Cyclodextrin (CD) Polymer

Five (5) 9 μL aliquots (total of 45 μL) of 3 mM thiolated CD polymer ofExample 23 was added to 20 μL of plasmid DNA (0.24 μg/μL) at 10 minuteintervals. The resulting solution was allowed to oxidize in airovernight. Electron microscopy showed the resulting supramolecularcomplex to be uniform in size.

Example 25 Oxidation of Thiolated Cyclodextrin (CD) Polymer withAldrithiol

Five (5) 9 μL aliquots (total of 45 μL of 3 mM thiolated CD polymer ofExample 23 was added to 20 μL of plasmid DNA (0.24 μg/μL) at 10 minuteintervals. Two equivalents of oxidizing reagent ALDRITHIOL (commerciallyavailable from Aldrich Chemical Company, Inc., Milwaukee, Wis.) based onthe thiolated CD polymer was immediately added to the solution andgently mixed by pipetting. Electron microscopy showed the resultingsupramolecular complex to be uniform in size.

Example 26 Synthesis of β-cyclodextrin(cystamine)-DMA copolymer, 16

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 180 mg (0.131 mmol) of 13 and 32 mg of dimethyl adipimidate (DMA,Pierce Chemical Co. of Rockford, Ill.). To this was added 500 μL of 0.5M Na₂CO₃. The resulting solution was covered with foil and stirredovernight. The mixture was acidified with 0.1 N HCl and dialyzed withSpectrapor MWCO 3,500 membrane for 2 days and lyophilized to afford 41mg of a white amorphous solid with Mw=14 kD, as determined by lightscattering.

Example 27 Synthesis of β-cyclodextrin(cystamine)-DMP copolymer, 17

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 160 mg (0.116 mmol) of 13 and 30.1 mg of dimethyl pimelimidate(DMP, Pierce Chemical Co. of Rockford, Ill.). To this was added 500 μlof 0.5 M Na₂CO₃. The resulting solution was covered with foil andstirred overnight. The mixture was then acidified with 0.1 N HCl anddialyzed with Spectrapor MWCO 3,500 membrane for 2 days and lyophilizedto afford 22 mg of a white amorphous solid with Mw=14 kD, as determinedby light scattering.

Example 28 Transfection Studies with Plasmids Encoding LuciferaseReporter Gene

BHK-21 cells were plated in 24-well plates at cell density of 60,000cells/well in 1 mL media. Plasmids encoding the luciferase gene weremixed with the CD-polymer as in Example 16 using copolymers 14, 15, 16,or 17. After 24 hours, media was removed and transfection mixture (200μL of Optimem with 20 μL of polymer/DNA solution) was added to eachwell. After 5 hours, 800 μL of complete media (DMEM+10% BS, Gibco) wasadded to each well. 24 hours after transfection, media was replaced with1 mL complete media. After another 24 hours, media was removed and cellswere washed with 1 mL PBS. Cells were then lysed with 0.050 mL of CellCulture Lysis Buffer (Promega) by one freeze-thaw cycle. 4 μL of celllysate was used for luciferase assay, measured in terms of light unitsproduced, and 10 μL was used for Biorad's protein DC assay. Thetransfection and toxicity results are illustrated in FIGS. 2A, 2B, 2Cand 2D.

Example 29 Effect of Heparan Sulfate on PEI/DNA Particle

Various concentrations of linear polyethyleneimine (1PEI) 2 kD weremixed with DNA for 30 minutes. Heparan sulfate (75× of DNA) was added tothe PEI/DNA solution for 30 minutes. An agarose gel was run to examinethe results. At low PEI/DNA ratio, heparan sulfate was able to strip PEIaway from DNA. However, at a higher concentration of PEI, PEI remainedassociated with the DNA even with the addition of heparan sulfate.

Example 30 Crosslinking Experiment Using Branched Polyethyleneimine(bPEI) 25 kDa with Varying Concentration of Heparan Sulfate

10 μL (1 μg) of DNA and 100_, of polyethyleneimine (PEI, 1.41 mM, 5+/−charge ratio) was mixed together for 30 minutes. Then crosslinkerdithiobispropionimidate (DTBP) or dimethylsuperimidate (DMS) was addedto the DNA/PEI solution. After 90 minutes, different concentration ofheparan sulfate (HS) was added for competitive binding with PEI. Theagarose gel was run after 20 minutes to examine the effect ofcrosslinker on the binding of PEI to DNA. For 0.1−/+HS, HS could notbind to PEI to cause PEI to dissociate from DNA. Higher concentrationsof HS could dissociate PEI from DNA only in the absence of DTBP (orDMS). Thus, with the presence of crosslinker on PEI, PEI has a higheraffinity to DNA. However, at 3−/+HS, the concentration is high enoughsuch that HS dissociated PEI from DNA even with the presence ofcrosslinker.

Example 31 Crosslinking Experiment with Pentalysine Using CrosslinkerDTBP and Reducing Agent Tris(2-carboxyethyl)phosphine (TCEP)

Pentalysine was added to DNA for 15 minutes. Crosslinker DTBP was thenadded to the solution mixture for over 60 minutes. TCEP was added and anagarose gel was run after 30 minutes. Pentalysine itself was not strongenough to bind to the DNA. However, with the addition of crosslinkerDTBP, crosslinked pentalysine bound to the DNA. When reducing agent TCEPwas added, pentalysine once again dissociated from DNA. Thus,crosslinking with DTBP increased the affinity of pentalysine to DNA.

Example 32 Reversible Crosslinking of Branched PEI (bPEI) (25 kDa) withDTBP

1 ug of DNA plasmid (˜5 kpb) was complexed with bPEI (25 kDa) at a 5+/−charge ratio for 30 minutes. Crosslinker DTBP (dithiobispropionimidate,Pierce Chemical Co. of Rockford, Ill.) was then added and allowed toreact with the primary amines on PEI for 90 minutes. After the reaction,some of the solutions were treated with a reducing agent, TCEP(Tris2-carboxyethyl)phosphine) for 25 minutes. Heparan sulfate was thenadded to the mixture at a 2:1 charge ratio with respect to PEI todissociate the particles.

Heparan sulfate was unable to dissociate crosslinked PEI from the DNA.However, after reduction of the crosslinking agent with TCEP, heparansulfate was able to dissociate the PEI from DNA. Thus crosslinking DTBPis able to stabilize PEI/DNA composites. This stabilization isreversible under reductive conditions. The results are illustrated inthe agarose gel of FIG. 1.

Example 33 β-Cyclodextrin(cystamine)-PEG600 Copolymer, 18

A 100 mL round-bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.564 g (1.25 mmol) of 13 and 25mL of freshly distilled dimethylacetamide (DMAc, Aldrich). To the slurrywas added 0.7 mL (4 eq) of triethylamine (Aldrich) and a solution of 10in 5 mL of DMAc. The resulting mixture was heated to 80° C. for 2 hours.After this time, the reaction was allowed to cool to ambient temperatureand the solvent was removed in vacuo. The residue was then taken up into50 mL of water and the resulting solution dialyzed against water in aSpectra/Por 7 MWCO 3,500 membrane. The resulting solution waslyophilized to dryness to afford 1.515 g (66%) of an off-white amorphoussolid with Mw=25,000, as determined by light scattering.

Example 34 Synthesis of β-Cyclodextrin-Tosylate, 19 (Melton, L. D., andSlessor, K. N., Carbohydrate Research, 18, p 29 (1971))

A 500 mL round-bottom flask equipped with a magnetic stirbar, a vacuumadapter and a septum was charged with a solution of dry β-cyclodextrin(8.530 g, 7.51 mmol) and 200 mL of dry pyridine. The solution was cooledto 0° C. before 1.29 g (6.76 mmol) of tosyl chloride was added. Theresulting solution was allowed to warm to room temperature overnight.The pyridine was removed as much as possible in vacuo. The resultingresidue was then recrystallized twice from 40 mL of hot water to yield7.54 (88%) of a white crystalline solid.

Example 35 Synthesis of β-cyclodextrin-thiol, 20 (K. Fujita, et al.,Bioorg. Chem., Vol. 11, p 72 (1982) and K. Fujita, et al., Bioorg.Chem., Vol. 11, p. 108 (1982))

A 50 mL round bottom flask with a magnetic stirbar and a Schlenk adapterwas charged with 1.00 g (0.776 mmol) of 19, 0.59 g (7.75 mmol) ofthiourea (Aldrich) and 7.8 mL of 0.1N NaOH solution. The resultingmixture was heated at 80° C. for 6 hours under nitrogen. Next, 0.62 g(15.5 mmol) of sodium hydroxide was added and the reaction mixture washeated at 80° C. under nitrogen for another hour. The reaction wasallowed to cool to room temperature before it was brought to pH 4.0 with10% HCl. The total solution volume was brought to 20 mL and then wascooled in an ice bath before 0.8 mL of tetrachloroethylene was added.The reaction mixture was stirred vigorously at 0° C. for 0.5 h beforethe precipitated solid was collected in a fine glass frit. The solid waspumped down overnight to yield 0.60 g (67%) of a white amorphous solid.

Example 36 Synthesis of β-cyclodextrin-iodide, 21

A round bottom flask with a magnetic stirbar and a Schlenk adapter ischarged with 19, 15 equivalents of potassium iodide, and DMF. Theresulting mixture is heated at 80° C. for 3 hours, after which thereaction is allowed to cool to room temperature. The mixture is thenfiltered to remove the precipitate and the filtrate evaporated todryness and redissolved in water at 0° C. Tetrachloroethylene is addedand the resulting slurry stirred vigorously at 0° C. for 20 minutes. Thesolid is collected on a medium glass frit, triturated with acetone andstored over P₂O₅.

Example 37 Synthesis of β-cyclodextrin-thiol-PEG Appended Polymer, 22

A 100 mL round-bottom flask equipped with a magnetic stirbar and areflux condensor was charged with 2.433 g (2.11 mmol) of 20 and 0.650 gof functionalized PEG (PEG with pendant olefins, received from YoshiyukiKoyama of Otsuma Women's University, Tokyo, Japan). The resultingmixture was heated at reflux for 12 hours, during which time 20dissolved. The reaction mixture was allowed to cool to room temperatureand precipitated solid was removed by centrifugation. The supernatantwas dialyzed against water in a Spectra/Por 7 MWCO 1,000 membrane. Thesolution was lyophilized to give an amorphous white solid.

Example 38 Synthesis of Branched PEI-cyclodextrin polymer, 23

A 20 mL scintillation vial equipped with a magnetic stirbar is chargedwith branched PEI (25 kD, Aldrich) and 22. To this is added degassedsodium carbonate buffer. The resulting solution stirred at 80° C. for 4hours. The mixture is acidified with 0.1 N HCl and dialyzed withSpectra/Por MWCO 3,500 membrane for 2 days and lyophilized.

Example 39 Synthesis of hexamethylenediamine-DMS copolymer, 24

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 80 mg (0.690 mmol) of hexamethylenediamine (Aldrich) and 195 mg ofdimethyl suberimidate (DMS, Pierce Chemical Co. of Rockford, Ill.). Tothis was added 250 μA of 0.5 M Na₂CO₃. The resulting solution wascovered with foil and stirred overnight. The mixture was then acidifiedwith 0.1 N HCl and dialyzed with Spectrapor MWCO 3,500 membrane for 2days and lyophilized to afford 30 mg of a white amorphous solid.

Example 40 Synthesis of 1,9-diaminononane-DMS copolymer, 25

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 85 mg (0.537 mmol) of 1,9-diaminononane (Aldrich) and 146 mg ofdimethyl suberimidate (DMS, Pierce Chemical Co. of Rockford, Ill.). Tothis was added 250 μA of 0.5 M Na₂CO₃. The resulting solution wascovered with foil and stirred overnight. The mixture was then acidifiedwith 0.1 N HCl and dialyzed with Spectrapor MWCO 3,500 membrane for 2days and lyophilized to afford 41.7 mg of a white amorphous solid.

Example 41 Transfections with Diamine-DMS Copolymers 24 and 25

Transfections were conducted as described in Example 26, exceptcopolymers 24 and 25 replaced copolymers 14, 15, 16, and 17.Transfection and toxicity results are illustrated in

FIG. 3A and FIG. 3B, respectively. Removal of cyclodextrin from thepolymer backbone results in polymers with high toxicity.

Example 42 Solubilization of Taxol with 18

Excess amounts of paclitaxel was added to an 18% solution of 18. Thesolutions were agitated, vortexed, and then filtered by a 2 μM nylonfilter to remove any undissolved paclitaxel. The filtered solution wasthen injected into an HPLC equipped with an Altima C8 reverse phasecolumn. Paclitaxel was detected by UV absorption at 227 nm, andconcentration of paclitaxel determined by peak integration. Calibrationplots of paclitaxel concentration vs. peak area showed a linearrelationship up to 25 μg/mL. The presence of 18% solution of 18 clearlyenhanced solubility of paclitaxel greater than 30 times.

Example 43 Delivery of Paclitaxel with 18 or 22

Cells are counted on a hemocytometer and plated at a density of 4,000cells/well in 96 well plates. Paclitaxel is mixed with polymer 18conjugated with a ligand or polymer 22 conjugated with a ligand fortargeted delivery. The solutions are allowed to mix for at least 30minutes, after which, the drug/polymer solutions are added to the cellswith serial dilution. The culture plates are incubated at 37° C. Aftertwo days, the IC₅₀ of the paclitaxel to the cells is determined by MTTassay. The culture medium is removed, and the cells are washed with PBS.Next, 50 μL/well MTT is added, followed by 150 μL/well media. After 4hours of incubation at 37° C., the MTT solution is removed and theformazan is solubilized by the addition of 200 μL/well DMSO. Theabsorbance of the formazan is read at 562 nm by a microtiter platereader.

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It will be apparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference in their entirety.

1. A method of preparing a supramolecular complex comprising the stepsof: contacting at least one therapeutic agent and at least one polymerto form a composite, and treating said polymer of said composite underconditions sufficient to form a supramolecular complex comprising saidtherapeutic agent and a multi-dimensional polymer network.
 2. The methodof claim 1, wherein said polymer of said composite is a substantiallylinear polymer, a substantially branched polymer, or a mixture thereof.3. The method of claim 2, wherein said substantially linear polymer islinear polyethyleneimine or a linear cyclodextrin-containing polymer andsaid substantially branched polymer is branched polyethyleneimine or abranched cyclodextrin-containing polymer.
 4. The method of claim 3,wherein said linear cyclodextrin-containing polymer is a linearcyclodextrin copolymer or a linear oxidized cyclodextrin copolymer andsaid branched cyclodextrin-containing polymer is a branched cyclodextrincopolymer or a branched oxidized cyclodextrin copolymer.
 5. The methodof claim 1, wherein said therapeutic agent is selected from the groupconsisting of an antibiotic, a steroid, a polynucleotide, a plasmid, apeptide, a peptide fragment, a small molecule, a chelating agent and abiologically active macromolecule.
 6. The method of claim 5, whereinsaid therapeutic agent is DNA.
 7. The method of claim 5, wherein saidtherapeutic agent is modified with at least one ligand.
 8. The method ofclaim 7, wherein said ligand is selected from the group consisting ofvitamins, proteins, and polysaccharides.
 9. The method of claim 1,wherein said treating step comprises the addition of at least onecrosslinking agent.
 10. The method of claim 9, wherein said crosslinkingagent is selected from the group consisting of adipic acid dihydrazide,polyethylene glycol 600 dihydrazide, dimethyl3,3′-dithiobispropionimidate, dithiobis(succinimidyl propionate),disuccinimidyl suberate, and dimethylsuberimidate (DMS).
 11. The methodof claim 1, further comprising the step of contacting said compositewith at least one ligand to form a ligand-containing composite.
 12. Themethod of claim 11, wherein said ligand is selected from the groupconsisting of vitamins, proteins, and polysaccharides.
 13. The method ofclaim 1, further comprising the step of contacting said supramolecularcomplex with at least one ligand to form a ligand-containingsupramolecular complex.
 14. The method of claim 13, wherein said ligandis selected from the group consisting of vitamins, proteins, andpolysaccharides.
 15. A supramolecular complex comprising: a compositecomprising at least one therapeutic agent and a polymer; and amulti-dimensional polymer network.
 16. A method of treating a subject,the method comprising the step of administering a therapeuticallyeffective amount of a supramolecular complex, wherein the supramolecularcomplex comprises: a composite comprising at least one therapeutic agentand a polymer; and a multi-dimensional polymer network.
 17. (canceled)18. (canceled)
 19. The supramolecular complex of claim 15 comprising aplurality of therapeutic agents.
 20. A method of delivering atherapeutic agent to a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of asupramolecular complex, wherein the supramolecular complex comprises: acomposite comprising at least one therapeutic agent and a polymer; and amulti-dimensional polymer network.
 21. The method of claim 16, whereinsaid therapeutic agent is selected from the group consisting of anantibiotic, a steroid, a polynucleotide, a plasmid, a peptide, a peptidefragment, a small molecule, a chelating agent and a biologically activemacromolecule.
 22. The supramolecular complex of claim 19, wherein saidtherapeutic agent is selected from the group consisting of anantibiotic, a steroid, a polynucleotide, a plasmid, a peptide, a peptidefragment, a small molecule, a chelating agent and a biologically activemacromolecule.
 23. The method of claim 20, wherein said therapeuticagent is selected from the group consisting of an antibiotic, a steroid,a polynucleotide, a plasmid, a peptide, a peptide fragment, a smallmolecule, a chelating agent and a biologically active macromolecule. 24.The method of claim 16, wherein said polymer of said composite is asubstantially linear polymer, a substantially branched polymer, or amixture thereof.
 25. The supramolecular complex of claim 15, whereinsaid polymer of said composite is a substantially linear polymer, asubstantially branched polymer, or a mixture thereof.
 26. The method ofclaim 20, wherein said polymer of said composite is a substantiallylinear polymer, a substantially branched polymer, or a mixture thereof.